The present invention is directed to methods for forming deoxynucleosides from their corresponding ribonucleosides by first forming tert-butylphenoxythiocarbonyl derivatives and subsequently effecting a radical deoxygenation reaction at the carbon attached to the site of the tert-butylphenoxythiocarbonyl group.
The potential therapeutic use of oligonucleotides represents a new paradigm for novel drug discovery. Over the last decade, oligonucleotide based antisense, triplex, ribozyme and aptamer techniques have emerged as powerful tools in the discovery of more specific and effective drugs (Sanghvi, Y. S., In Comprehensive Natural Product Chemistry; Barton, D. H.; Nakanishi, K. (ed. in chief); vol. 7: DNA and Aspects of Molecular Biology; Kool. E. (ed.); Pergamon: New York, 1999, 285). Among these techniques, the antisense approach leads the trend with over a dozen oligonucleotides currently undergoing human clinical trials for the treatment of viral infections, cancers, and inflammatory disorders. For example, ISIS 2922 (formivirsen sodium) is a 21 mer antisense phosphorothioate that inhibits the replication of the human cytomegalovirun (HCMV). The recent success of antisense drugs in clinical trials is creating a growing demand for the manufacture of oligonucleotides.
Advances in automated synthesis on solid support and commercialization of synthetic nucleic acid building blocks now allows the generation and screening of an unprecedented number of synthetic oligonucleotides. Oligonucleotides are synthesized on automated DNA/RNA synthesizers with nucleoside phosphoramidites employed as the most commonly used monomers. Nucleoside phosphoramidites can be produced from the phosphitylation of 5xe2x80x2-dimethoxytrityl protected 2xe2x80x2-deoxynucleosides.
For the commercialization of antisense drugs, consumption of large amounts of 2xe2x80x2-deoxynucleosides are necessary. 2xe2x80x2-deoxynucleosides currently originate from natural sources, especially from salmon fish milt. The worldwide output of fish milt is about twenty thousand tons per year. From this, only about one hundred tons of DNA salt can be generated. The DNA salt is degraded to give approximately ten tons of 2xe2x80x2-deoxynucleosides in an even distribution of the four 2xe2x80x2-deoxynucleosides (dA, dC, dG, and T). A maximum of one ton of oligonucleotides can be produced from ten tons of 2xe2x80x2-deoxynucleosides, assuming that all ten tons would be available for oligonucleotide production.
According to our predictions, the market for the first three antisense drugs alone, not to mention the market for oligonucleotides used as other types of drugs and as diagnostic reagents, will require at least one ton of oligonucleotides, indicating that natural resources are insufficient to provide enough 2xe2x80x2-deoxynucleosides to meet future antisense drug demand. In addition, due to declining fish stock, fish milt may be an unreliable source of 2xe2x80x2-deoxynucleotides. Because the demand for 2xe2x80x2-deoxynucleosides exceeds the supply to such a great extent, a need exists for alternative sources of 2xe2x80x2-deoxynucleosides.
The supply of RNA and ribonucleosides is much greater than deoxynucleosides. RNA is derived from yeast and ribonucleosides can be produced in large amounts by fermentation processes. Due to their increased availability, ribonucleosides are much less expensive than 2xe2x80x2-deoxynucleosides. Methods exist for synthetically deriving deoxynucleosides from their ribonucleoside counterparts. Nevertheless, these methods are not economically feasible for the large scale production of 2xe2x80x2-deoxynucleosides. For example, ribonucleotides in their 5xe2x80x2-di or triphosphate form can be biosynthetically converted to their 2xe2x80x2-deoxy counterparts by ribonucleotide reductases. However, these processes are undesirable due to multiple inherent difficulties in the scaled-up production of 2xe2x80x2-deoxynucleosides catalyzed by these reductases.
Other possibilities exist for deriving deoxynucleosides from ribonucleosides. For instance, the chemical transformations used for converting alcohol groups to their corresponding deoxy derivatives are viable options. This chemistry involves radical chain reactions wherein thiocarbonyl derivatives of the alcohol groups are deoxygenated using free radical initiators and tributyltin hydride, as described by Barton and McCombie (Barton, D. H. R.; McCombie, S. J., J. Chem. Soc., Perkin Trans. I, 1975, 1574). These reactions are useful for the 2xe2x80x2-deoxygenation of ribonucleosides as well. (Robins, M. J.; Wilson, J. S., J. Am. Chem. Soc., 1981, 103, 932, Robins, M. J.; Wilson, J. S.; Hansske, F., J. Am. Chem. Soc., 1983, 105, 4059). Robins developed a thiocarbonyl reagent, phenyl chlorothionoformate (PhOCSCl, $44.75/5 g, Aldrich(trademark) 1998-1999), that is introduced onto the 2xe2x80x2position of a ribonucleoside by a simple acylation. Chemical 2xe2x80x2-deoxygenation of the 2xe2x80x2-thiocarbonyl ribonucleoside is subsequently effected by a radical reaction. In addition to having a higher cost associated with the reagents these reactions use tin reagents for reductions which are toxic and difficult to dispose of.
The method developed by Robins was improved when the phenyl groups of the thiocarbonyl reagents were substituted with electron donating groups, such as halogens. (Barton, D. H.; Jaszberenyi, J. C., Tetrahedron Letters, 1989, 30, 2619, Barton, D. H. R.; Dorchak, J.; Jaszberenyi, J. C., Tetrahedron Letters, 1992, 36, 7435). Barton found that substituted phenyl chlorothionoformates, such as, 2,4,6-trichlorophenyl chlorothionoformate ($58.70/5 g Aldrich(trademark) 1998-1999), or especially when pentafluorophenyl chlorothionoformate ($64.00/5 g Aldrich(trademark) 1998-1999) is used to make the thiocarbonyl derivative, radical deoxygenation reaction rates with tributyltin hydride are considerably increased, occurring in minutes rather than hours. Additionally, the yields were found to be excellent. The electron withdrawing inductive effect of the substituents increases the radicophilicity of the thiocarbonyl group, thereby speeding up reaction rates. Although this method may be effective for the large scale production of 2xe2x80x2-deoxynucleosides from their corresponding ribonucleosides, the cost of the substituted phenylthiocarbonyl compounds is prohibitively high.
The use of a series of substituted 3xe2x80x2-phenyl thionocarbonates has been described wherein a free radical coupling using oximes mediated by bis(trimethylstannyl)-benzopinacolate. These reactions led to the formation of carbon-carbon bonds in the preparation of a series of dimeric nucleosides as mimics of nucleic acids (Bhat, B.; Swayze, E. E.; Wheeler, P.; Dimock, S.; Perbost, M.; Sanghvi, Y., J. Org. Chem., 1996, 61, 8186, Dimock S.; Bhat, B.; Peoc""h, D.; Sanghvi, Y. S.; Swayze, E. E., Nucleosides and Nucleotides, 1997, 16(7-9) 1629).
The present invention addresses the need for cost-effective methods for the large-scale production of 2xe2x80x2-deoxynucleosides from their corresponding ribonucleosides.
The present invention provides processes for preparing a 2xe2x80x2-deoxynucleoside comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form a 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside;
contacting the 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 2xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the bisprotected ribonucleoside; and
treating the derivatives with a reducing agent for a time and under conditions effective to give the 2xe2x80x2-deoxynucleoside.
Preferred protecting agents include 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane.
A preferred reducing agent will convert the hydroxy to the hydrogen by a radical deoxygenation step.
According to one aspect of the present invention, a process for preparing a 2xe2x80x2-deoxynucleoside comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form a 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside;
contacting the 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 2xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the bisprotected ribonucleoside; and
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to form the 2xe2x80x2-deoxyribonucleoside.
In one aspect of the invention radical reagents include tributyltin hydride, solid supported tributyltin hydride, triethylsilyl hydride, a poly(alkyl)hydrosiloxane, or poly(methyl)hydrosiloxane.
In another aspect of the present invention a process for generating a 2xe2x80x2-deoxynucleoside radical is provided comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form the 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside;
contacting the 3xe2x80x2-O,5xe2x80x2-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form the isomeric 2xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the bisprotected ribonucleoside; and
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to form the corresponding 2xe2x80x2-deoxynucleoside radical.
In a further aspect of the present invention, a process for making a 2xe2x80x2,3xe2x80x2-dideoxynucleoside is provided comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with a first protecting agent for a time and under conditions effective to give a 5xe2x80x2-O-protected ribonucleoside;
treating the 5xe2x80x2-O-protected ribonucleoside with a second protecting agent for a time and under conditions effective to give a 2xe2x80x2-O,5xe2x80x2-O-protected ribonucleoside;
treating the 2xe2x80x2-O,5xe2x80x2-O-protected ribonucleoside with an acylating agent for a time and under conditions to give a 2xe2x80x2-O-protected-3xe2x80x2-O-acyl-5xe2x80x2-O-protected ribonucleoside;
treating the 2xe2x80x2-O-protected-3xe2x80x2-O-acyl-5xe2x80x2-O-protected ribonucleoside with a first deprotecting agent for a time and under conditions effective to give a 3xe2x80x2-O-acyl-5xe2x80x2-O-protected ribonucleoside;
contacting the 3xe2x80x2-O-acyl-5xe2x80x2-O-protected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 2xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the 3xe2x80x2-O-acyl-5xe2x80x2-O-protected ribonucleoside;
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to form a 3xe2x80x2-O-acyl-5xe2x80x2-O-protected-2xe2x80x2-deoxynucleoside radical;
subjecting the 3xe2x80x2-O-acyl-5xe2x80x2-O-protected-2xe2x80x2-deoxynucleoside radical to conditions effective to eliminate the 3xe2x80x2-O-acyl group thereby forming a 5xe2x80x2-O-protected-2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxynucleoside;
optionally treating the 5xe2x80x2-O-protected-2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxynucleoside with a second deprotecting agent for a time and under conditions effective give a 2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxynucleoside; and
reducing the optionally deprotected 2xe2x80x2,3xe2x80x2-didehydro-2xe2x80x2,3xe2x80x2-dideoxynucleoside to give the 2xe2x80x2,3xe2x80x2-dideoxynucleoside.
In a preferred embodiment the 5xe2x80x2-O-protecting group is acid-labile with trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX) being preferred.
Preferred conditions to effect elimination of the 3xe2x80x2-O-acyl group include at least one of exposure to light, heating and treatment with at least one chemical reagent.
A preferred 3xe2x80x2-O-acyl group has the formula:
3xe2x80x2xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R
wherein R is substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted aryl having 6 to about 14 carbon atoms, wherein the substituent groups are selected from alkyl, aryl, alkoxy, carboxy, benzyl, phenyl, halogen, alkenyl and alkynyl. A preferred R group is R is C1-C10 alkyl with CH3 being more preferred.
In another aspect of the present invention, a process for preparing a 2xe2x80x2,3xe2x80x2-dideoxynucleoside is provided comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with a protecting agent for a time and under conditions effective to give a 5xe2x80x2-O-protected ribonucleoside;
contacting the 5xe2x80x2-O-protected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 2xe2x80x2,3xe2x80x2-O-bis-tert-butylphenoxythiocarbonyl derivatives of the 5xe2x80x2-O-protected ribonucleoside; and
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to give the 2xe2x80x2,3xe2x80x2-dideoxynucleoside.
In one aspect of the present invention a process for preparing a 5xe2x80x2-deoxynucleoside is provided comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form a 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside;
contacting the 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 5xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the bisprotected ribonucleoside; and
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to give the 5xe2x80x2-deoxynucleoside.
In a further embodiment transient protection of the 5xe2x80x2-hydroxyl position of the ribonucleoside is effected by treating the ribonucleoside with a labile protecting agent effective to protect the 5xe2x80x2-hydroxyl position prior to forming the 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside allowing selective removal of the 5xe2x80x2-protecting group by treatment with a deprotecting agent subsequent to formation of the 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside.
In a preferred embodiment the 5xe2x80x2-deoxynucleoside is formed by radical deoxygenation.
In yet another aspect of the present invention a process for preparing a 5xe2x80x2-deoxynucleoside comprising the steps of:
selecting a ribonucleoside;
treating the ribonucleoside with at least one protecting agent for a time and under conditions effective to form a 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside;
contacting the 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form isomeric 5xe2x80x2-O-tert-butylphenoxythiocarbonyl derivatives of the 2xe2x80x2-O,3xe2x80x2-O-bisprotected ribonucleoside; and
treating the derivatives with a radical reagent and a radical initiator for a time and under conditions effective to form the corresponding 5xe2x80x2-deoxyribonucleoside.
In a preferred embodiment the radical reagent is tributyltin hydride, solid supported tributyltin hydride, triethylsilyl hydride, a poly(alkyl)hydrosiloxane or poly(methyl)hydrosiloxane.
In a further aspect of the present invention a process for converting a hydroxyl group to hydrogen comprising the steps of:
selecting a compound having the hydroxyl group;
contacting the compound with an isomeric mixture of tert-butylphenyl chlorothionoformates, preferably comprising from about 87% to about 99% 3-tert-butylphenyl chlorothionoformate and from about 1% to about 13% 4-tert-butylphenyl chlorothionoformate, for a time and under conditions effective to form a mixture of isomeric tert-butylphenoxythiocarbonyl derivatives of the compound; and
treating the derivatives of the compound with a reducing agent for a time and under conditions effective to convert the hydroxyl group of the compound to hydrogen.
In preferred embodiments, the reducing step is effected by treating the derivatives with a radical initiator and a radical reagent to effectuate a radical deoxygenation reaction. Preferred radical initiators include azo initiators such as for example: AIBN (2,2xe2x80x2-azobisisobutyro-nitrile), ACN (VASO(trademark); 1,1xe2x80x2-azobis[cyclohexanecarbo-nitrile]); diacyl peroxide initiators: benzoyl peroxide (dibenzoyl peroxide), and ultraviolet light; and polymerization initiators including for example VA-044, V-50, VA-061, V-501, VA-086, V-70, V-65B, V-601, V-59, and V-40. Preferred radical reagents include tributylin hydride, solid supported tributylin hydride, solid supported triethylsilyl hydride, and poly(alkyl)hydrosiloxane, such as poly(methyl)hydrosiloxane.